CTX-M ESBL-producing Enterobacteriaceae: estimated prevalence in adults in England in 2014

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1 J Antimicrob Chemother 2018; 73: doi: /jac/dky007 Advance Access publication 5 March 2018 CTX-M ESBL-producing Enterobacteriaceae: estimated prevalence in adults in England in 2014 Cliodna A. M. McNulty 1 *, Donna M. Lecky 1, Li Xu-McCrae 2, Deborah Nakiboneka-Ssenabulya 1, Keun-Taik Chung 3, Tom Nichols 4, Helen Lucy Thomas 4, Mike Thomas 5, Adela Alvarez-Buylla 2, Kim Turner 1, Sahida Shabir 2, Susan Manzoor 2, Stephen Smith 6, Linda Crocker 1 and Peter M. Hawkey 3 1 Public Health England, Primary Care Unit, Microbiology Department, Gloucester Royal Hospital, Great Western Road, Gloucester GL1 3NN, UK; 2 Public Health England, Heart of England NHS Foundation Trust, Public Health Laboratory, Bordesley Green E, Birmingham B9 5SS, UK; 3 Institute of Microbiology and Infection, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK; 4 Public Health England, 61 Colindale Avenue, London NW9 5EQ, UK; 5 University of Southampton, Aldermoor Health Centre, Aldermoor Close, Southampton SO16 5ST, UK; 6 University Hospitals of Coventry & Warwickshire NHS Trust, Midlands & NW Bowel Cancer Screening Hub, Hospital of St Cross, Barby Road, Rugby CV22 5PX, UK *Corresponding author. Tel:! ; cliodna.mcnulty@phe.gov.uk Received 2 June 2017; returned 3 September 2017; revised 14 December 2017; accepted 2 January 2018 Background: ESBL-producing Enterobacteriaceae (ESBLPE) are increasing in prevalence worldwide and are more difficult to treat than non-esblpe. Their prevalence in the UK general population is unknown, as the only previous UK ESBLPE faecal colonization study involved patients with diarrhoea. Objectives: To estimate the prevalence of CTX-M ESBLPE faecal colonization in the general adult population of England in 2014, and investigate risk factors. Methods: A stratified random sample of registered patients from 16 general practices within four areas of England were invited to participate by returning faeces and self-completed questionnaires. Specimens were tested for ESBLPE and carbapenemase-producing Enterobacteriaceae (CPE). Results: 2430 individuals participated (4% of those invited). The estimated prevalence of colonization with CTX-M ESBLPE in England was 7.3% (95% CI 5.6% 9.4%) (Shropshire 774 participants, 4.9% colonization; Southampton City 740 participants, 9.2%; Newham 612 participants, 12.7%; Heart of Birmingham 234 individuals, 16.0%) and was particularly high in: those born in Afghanistan (10 participants, 60.0% colonization, 95% CI 29.7% 84.2%); those born on the Indian subcontinent (India, Pakistan, Bangladesh or Sri Lanka) (259 participants, 25.0% colonization, 95% CI 18.5% 32.9%); travellers to South Asia (India, Pakistan, Bangladesh, Sri Lanka or Nepal) in the last year (140 participants, 38.5% colonization, 95% CI 27.8% 50.5%); and healthcare domestics (8 participants, unweighted 37.5% colonization, 95% CI 8.5% 75.5%). Risk factors identified included: being born in the Indian subcontinent (aor 5.4, 95% CI ); travel to South Asia (aor 2.9, 95% CI ) or to Africa, China, South or Central America, South East or Pacific Asia or Afghanistan (aor 2.6, 95% CI ) in the last year; and working as a healthcare domestic (aor 6.2, 95% CI ). None of the 48 participants who took co-amoxiclav in the last year was colonized with CTX-M ESBLPE. -15 accounted for 66% of CTX-M ESBLPE positives. 0.1% (two participants) were colonized with CPE. Conclusions: CTX-M ESBLPE are established in the general population in England and prevalence is particularly high in people from certain countries of birth or with recent travel. We recommend that these findings be taken into account in guidance on the empirical management of patients presenting with a likely Enterobacteriaceae infection. Introduction Extensive overuse of antibiotics worldwide has led to increasing prevalence of antibiotic-resistant Gram-negative bacteria (mainly Escherichia coli) that produce ESBLs; 85% 90% of these being CTX-M genotypes. 1 4 Carriage is particularly high in South Asia. 4 Between 2010 and 2015 total E. coli bloodstream and urine VC The Author(s) Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com 1368

2 CTX-M ESBL-producing Enterobacteriaceae prevalence JAC infections in England have continued to rise and, moreover, resistance in E. coli to important hospital antibiotics such as coamoxiclav and piperacillin/tazobactam rose significantly making treatment more difficult. 5,6 ESBL-producing Enterobacteriaceae (ESBLPE) are opportunistic pathogens with large bowel colonization typically preceding an ESBLPE infection, 7,8 so we believe that understanding the prevalence of faecal colonization overall and for certain sections of the general population will help inform empirical antibiotic guidance. Recent European studies indicate that travellers to countries outside Europe have an up to 10-fold higher prevalence of ESBLPE faecal colonization than the local population. 3,9,10 The prevalence of CTX-M ESBLPE in diagnostic faecal in a UK laboratory in 2010 was double in Middle Eastern/South Asian patients (22.8%) compared with Europeans (8.1%). 11 Studies in returning European travellers have found that travel to South Asia was the most important risk factor, 12 while antibiotic use 10,12,13 and travellers diarrhoea were other possible risk factors for ESBLPE acquisition. Travel acquisition is important as between 1% and 8% of returning travellers are hospitalized, 14,15 equating to million Europeans each year. 16 Importantly, there are no studies of the colonization of ESBLPE in the UK general population. This study aimed to estimate the prevalence of colonization with CTX-M ESBLPE across different sections of the adult general population of England in 2014, including different ethnic groups, and to investigate the potential risk factors for their carriage. Methods The study was undertaken in four NHS Primary Care Trusts (PCTs) in England. PCTs were state-funded and commissioned primary medical care from general practices in England until 2015; this has now been taken over by Clinical Commissioning Groups. All the UK population are registered with, or have access to, a general medical practice whom they consult for primary care. The four PCTs were purposively selected to capture the UK ethnic diversity: Newham (London, highest ethnic diversity), Heart of Birmingham (predominantly Asian), Shropshire (rural, mostly white British) and Southampton City (mixed ethnicity). Three to five willing Primary Care Research Network practices from each PCT were non-randomly selected to broadly represent each area with respect to ethnicity and deprivation. Individuals aged 18 years in selected practices were stratified by record of ethnicity (white, Asian, black, other/mixed or unknown), sex and age. Within each stratum individuals were randomly selected in 2013 and 2014 to receive an invitation letter. Respondents were sent study information, a faeces sample collection kit (not rectal swab), 5 incentive offer and a questionnaire (Figure S1, available as Supplementary data at JAC Online) (including questions on age, ethnic group, country of birth, employment, household characteristics, hospitalization, antibiotic use, travel abroad in past year and diet). Those not returning kits received a telephone reminder. Respondents were asked to collect scoops of faeces from both ends and the middle of their faeces sample and place them in a sterile container, keep the container cool and return by first-class post within 24 h to a central laboratory. As sampling progressed, faeces returns were monitored and invitations to reach Asian, black and younger age group sample sizes increased as necessary, including direct approach by general practice receptionists to individuals in some ethnic groups. For some practices all individuals within a given stratum were invited. Laboratory analysis Faecal samples were screened for ESBLPE, using direct culture on selective medium (Brilliance TM ESBL agar, Oxoid Ltd) for 24 h. To increase sensitivity, all samples were enriched as well as directly plated; 20 mg of each faecal sample was incubated for 24 h in 10 ml of brain heart infusion (BHI) broth containing 1 mg/l cefpodoxime 17 and subcultured onto Brilliance TM agar as before. 17,18 Oxidase-negative presumptive colonies of ESBLPE were defined as:!, 1 10 cfu;!!, cfu;and!!!, 100cfu.Onecolonyfrom each different colony morphology, from each plate of Brilliance agar, was identified using MALDI-TOF MS (Bruker UK Ltd) and tested for the gene using multiplex PCR. 19 Full-length gene amplification and sequencing identified genotypes (Table 1). A 10 lg ertapenem disc was placed on all selective plates to detect potential carbapenemase-producing Enterobacteriaceae (CPE). 20 Colonies growing in the zone of inhibition were tested by PCR for CPE genes. 21 Data analysis To estimate the prevalence of ESBLPE colonization for adults living in England in 2014 we used post-stratification weights based on the 2011 national census and number of eligible individuals at selected practices. To estimate the prevalence for each GP practice and PCT we used sampling weights based on the numbers of eligible individuals in each group at each practice. A new variable region of origin was derived mainly from ethnic group and country of birth. Multivariable logistic regression models were used to control for country of birth and region of origin (if born in the UK) (Table 2). Based on this preliminary analysis, factors that were associated with an increased risk of colonization were considered in further analysis. We also considered the strength of evidence and the number of missing values, and the evidence from other studies; we did not follow an automated model selection process. The final multivariable model for colonization with CTX-M ESBLPE included country of birth and region of origin (if born in the UK) as a factor variable with eight categories, the base category of which was born in some country not including the UK, India, Pakistan or Bangladesh (IPB), Sri Lanka, Afghanistan or the Middle East (Table 3). From the final model we estimated the adjusted ORs (aors) for each risk factor, the percentage of carriers attributable to each risk factor (for example, travel to India) and, for groups of risk factors (for example, travel abroad in past year), the population attributable fraction (PAF). The PAF is dependent on both the aor and the probability of being exposed to the factor. Among participants colonized with CTX-M ESBLPE we calculated the percentage colonized with a particular genotype, and the percentage belonging to particular ethnic groups among those who were carriers of a particular genotype. Ethics Approval for the study was obtained from the NRES Committee South West - Frenchay, Bristol, UK (13/SW/0017). The data we collected from GP practices were anonymous. Results Of adult individuals registered in 16 GP practices, were invited to participate; 3389 (5.8%) expressed interest and were sent a faeces kit, and 2331 (4.0%) returned a sample. A further 99 individuals invited by general practice receptionists participated, making a total of 2430 participants. The number of stool received from participants in each section of the adult population in England, the number of positive for ESBLPE, and the unweighted and weighted percentage positive for ESBLPE are shown in Table 2. The estimated prevalence of colonization with CTX-M ESBLPE in adults living in England in 2014 was 7.3% (95% CI 5.6% 9.4%). Of the four PCTs, Heart of Birmingham teaching PCT (234 participants) had the highest estimated prevalence at 16.0% (95% CI 1369

3 McNulty et al. Table 1. List of PCR and sequencing primers CTX-M group Name Primer sequence Location 8 and 25/ CTXM26 TTG ATT AAC TAC AAC CCC AT CTX-M-26 ( ) 3 0 CTXM26 GAT ATC ATT CGT CGT ACC AT CTX-M-26 ( ) 5 0 CTXMF26-1 CTC TGC GCA ATC TGA CGT TG CTX-M-26 ( ) 5 0 CTXMF26-2 AAG GCG GGC GAT GTT AAT GA CTX-M-26 (18 37) 3 0 CTXMR26-1 GCC AAT CGT ACG GGC AAA TG CTX-M-26 ( ) 1 ISEcp1 AAA AAT GAT TGA AAG GTG GT ISEcp1 (#149 to #128) 3 0 CTXM-1R ATA CAT CGC GAC GGC TTT CT CTX-M-1 ( ) 5 0 G1S1 ATG GTT AAA AAA TCA CTG CG CTX-M-15 (1 20) G9full GAA TAC TGA TGT AAC ACG GAT CTX-M-9 (#40 to #22) 3 0 G9full AGT TAC AGC CCT TCG GCG AT CTX-M-9 ( ) 10.2% 24.2%) [Newham 612 participants, 12.7% (95% CI 9.1% 17.4%); Southampton City 740 participants, 9.2% (95% CI 6.1% 13.7%); and Shropshire County 774 participants, 4.9% (95% CI 3.4% 7.0%)]. There was no evidence that estimated prevalence differed by age or sex. There were high estimated prevalences for participants born in India (136 participants, 28.7% prevalence, 95% CI 18.8% 41.2%), Pakistan (81 participants, 18.6% prevalence, 95% CI 10.5% 30.8%), Bangladesh (34 participants, 23.5% prevalence, 95% CI 11.8% 41.3%), Sri Lanka (8 participants, 25.0% prevalence, 95% CI 7.2% 59.0%), Afghanistan (10 participants, 60.0% prevalence, 95% CI 29.7% 84.2%) and the Middle East (18 participants, 15.5% prevalence, 95% CI 4.7% 40.5%) (Figure 1). The overall estimated prevalence for those born in the Indian subcontinent (India, Pakistan, Bangladesh or Sri Lanka) combined (259 participants) was 25.0%, (95% CI 18.5% 32.9%); differences between these four countries were non-significant (P " 0.65). The estimated prevalence for those born in the UK with an IPB region of origin was 15.7% (52 participants), while for those born in the UK with a UK region of origin (1459 participants) it was 5.6% (95% CI 4.4% 7.1%, P " 0.03). However there was a low estimated prevalence for those born in Africa and of the IPB ethnic group (17 participants, 0% prevalence, 95% CI 0% 19.5%). Overall estimated prevalence of ESBLPE was the same in those reporting taking any antibiotic in the last year (777 participants, 6.8% prevalence 95% CI 5.1% 8.9%) or not (1427 participants, 6.8% prevalence 95% CI 5.4% 8.6%). None of 48 participants who reported having taken any co-amoxiclav in the past 12 months carried ESBLPE (0% prevalence, 95% CI 0% 7.4%, P " 0.03). Estimated prevalence of ESBLPE in the 15 participants who had taken ciprofloxacin was 9.5%, (95% CI 2.5% 30.1%, P " 0.61). Two other groups with high estimated prevalence were those who had travelled to South Asia (India, Pakistan, Bangladesh, Sri Lanka or Nepal) in the last year (140 participants, 38.5% prevalence, 95% CI 27.8% 50.5%) and those working as a domestic in a healthcare setting [8 participants, unweighted prevalence 37.5%, 95% CI 8.5% 75.5% (3/8; 1 black African, CTX-M-15, no travel abroad; 1 white British, CTX-M-15, travelled to India with partner for 12 days; 1 Asian Indian, CTX-M-27, travelled to India alone for 42 days)]. If someone else in the participant s household had been to India, Bangladesh, the Middle East, the Indian subcontinent or South East or Pacific Asia, this significantly increased the participant s risk for carrying ESBL. Participants whose housemates had travelled abroad in the last year to the Indian subcontinent (India, Pakistan, Bangladesh or Sri Lanka) (120 participants, estimated prevalence 22.7%, 95% CI 14.2% 34.1%, P, 0.001), India (83 participants, estimated prevalence 24.9% 95% CI 14.5% 39.2%, P, 0.001) and the Middle East (43 participants, estimated prevalence 16.9%, 95% CI 7.5% 33.6%, P " 0.02) had a higher prevalence. In 1071 of 1234 participants, both the housemate and the main participant had travelled to the same country in the past 12 months. Relative frequency of CTX-M genotypes (Figure 2) Two hundred and eight participants were found to carry the CTX-M gene; 184 (88%) by direct culture (25%!, 26%!!, 37%!!!) and a further 24 (12%) on enrichment; no single participant had more than one species of bacteria carrying CTX-M. Most isolates (199) were E. coli, 5 Klebsiella pneumoniae, 4 Enterobacter and 1 Citrobacter. All isolates could be allocated a CTX-M grouping. Seventy-seven percent of participants with CTX-M ESBLPE (161/ 208) were colonized with CTX-M group 1; 25% (53/208) were colonized with CTX-M group 9; 4% (9/208) were colonized with both group 1 and group 9; and 1.4% (3/208) were colonized with CTX-M group 8, 25 or 26. Four isolates from group 1 and one isolate from group 9 could not be sequenced. The most common genotype was -15 (66%, 134/204) (Figure 2). Two other common genotypes were -14 (11%, 23/207) and -27 (13%, 27/207). CTX-M genotypes and risk factors Carriers of -15 had a similar probability of being of white ethnicity compared with carriers of other genotypes (62/134, 46% of -15 were white; 46/134, 34% Asian IPB; and 26/134, 19% other ethnicity) and 84/129 (65%) had travelled abroad in the last year. A carrier of -27 had a higher chance of being of the Asian-IPB ethnic group (16/27, 59% Asian IPB; 6/27, 22% white; 5/27, 19% other ethnicity; 16/27, 59% travelled abroad) compared with carriers of other genotypes. A -14 carrier had a higher chance of being of white ethnicity (19/23, 83% white; 2/23, 9% Asian IPB; 2/23, 9% other ethnicity; 14/23, 61% travelled abroad) compared with carriers of other genotypes. Carriers of the different genotypes CTX-M-15, -27 and -14 had a similar chance of having 1370

4 CTX-M ESBL-producing Enterobacteriaceae prevalence JAC Table 2. Prevalence of colonization with blactx-m ESBLPE in different sections of the adult population of England in 2014 Factor (unweighted %) Prevalence [95% CI] (weighted %) a Test for a difference in prevalence between the groups of each factor OR adjusted for b (country of birth and region born in the UK) (aor) [95% CI] Test for the effect of each factor after adjustment for country of birth and region born in the UK Overall [5.6, 9.4] Practice/Medical Centre Lathom Road [6.0, 19.6] Stratford Village [4.6, 17.0] 1.1 [0.5, 2.3] St Bartholomew s [9.3, 24.9] 1.6 [0.8, 3.3] Greet Medical Practice [5.6, 22.1] 1.7 [0.6, 4.4] High Trees [6.4, 43.5] 1.8 [0.7, 4.1] Khattak Memorial [8.9, 35.9] 1.2 [0.4, 3.6] Alma [6.0, 21.7] 1.3 [0.6, 2.9] Aldermoor [4.4, 10.5] 1.1 [0.5, 2.4] Church Stretton [3.8, 10.8] 1.2 [0.5, 2.8] Plas Ffynnon [2.2, 6.4] 0.7 [0.3, 1.7] The Caxton [2.4, 9.1] 0.8 [0.3, 2.1] Mulberry House [5.2, 16.8] 1.7 [0.7, 3.9] City Road [4.9, 39.7] 1.0 [0.3, 2.7] Nicholstown [9.7, 48.3] 1.0 [0.3, 2.7] Barking Road [6.2, 39.6] 1.4 [0.4, 5.4] St Deny s [1.2, 29.6] 1.8 [0.4, 8.8] PCT Newham [9.1, 17.4] Heart of Birmingham teaching [10.2, 24.2] 1.1 [0.7, 1.8] Shropshire County [3.4, 7.0] 0.7 [0.4, 1.2] Southampton City [6.1, 13.7] 1.0 [0.6, 1.5] Age group (years) [5.5, 10.5] 0.54 (test for (test for [3.5, 8.8] trend: P " 0.30) 1.1 [0.7, 1.8] trend: P " 0.77) [4.3, 9.8] 0.9 [0.6, 1.5] [5.2, 9.6] 1.1 [0.7, 1.7] [5.0, 10.5] 1.3 [0.8, 2.1] [1.6, 8.4] 0.8 [0.4, 1.8] Gender male [5.5, 9.5] [0.9, 1.6] 0.26 female [5.0, 7.8] 1 GP record of whether antibiotic used in the year before no [5.6, 8.6] yes [4.6, 8.1] 1.1 [0.8, 1.5] Continued 1371

5 McNulty et al. Table 2. Continued Factor (unweighted %) Prevalence [95% CI] (weighted %) a Test for a difference in prevalence between the groups of each factor OR adjusted for b (country of birth and region born in the UK) (aor) [95% CI] Born in the UK? UK [4.6, 7.4],0.001 other [8.9, 14.4] Country of birth UK [4.6, 7.4],0.001 Ireland [0.6, 24.5] India [18.8, 41.2] Pakistan [10.5, 30.8] Bangladesh [11.8, 41.3] Sri Lanka [7.2, 59.0] Nepal [0, 60.2] Afghanistan [29.7, 84.2] Africa [2.2, 14.5] Australasia [1.2, 48.0] Caribbean [3.7, 15.1] China [0, 19.5] Eastern Europe [0.6, 15.1] Middle East [4.7, 40.5] North America [1.0, 44.6] South or Central America [0, 24.7] South East or Pacific Asia [4.0, 35.6] Western Europe (excl. UK and Ireland) [1.5, 19.5] Mauritius [0, 30.8] Country of birth among the Indian, Pakistani or Bangladeshi (IPB) ethnic group Asian-IPB; born in UK [7.5, 39.1],0.001 Asian-IPB; born in India [19.2, 42.4] Asian-IPB; born in Pakistan [9.8, 30.2] Asian-IPB; born in Bangladesh [11.8, 41.3] Asian-IPB; born in Africa [0, 19.5] Asian-IPB; born in some other country [6.1, 78.9] not Asian-IPB [4.8, 7.2] Ethnic group c White-British [4.5, 7.2],0.001 White-Irish [0.5, 20.2] White-Gypsy or Irish Traveller [0, 84.2] White-Other [2.2, 11.1] Test for the effect of each factor after adjustment for country of birth and region born in the UK 1372

6 CTX-M ESBL-producing Enterobacteriaceae prevalence JAC Mixed-White and Black Caribbean [0, 45.9] Mixed-White and Black African [0, 45.9] Mixed-White and Asian [3.1, 38.6] Mixed-Other [0.6, 26.2] Asian-Indian [17.6, 36.8] Asian-Pakistani [10.2, 28.0] Asian-Bangladeshi [9.9, 36.3] Asian-Chinese [0, 14.8] Asian-Other [11.7, 33.6] Black-African [2.2, 11.3] Black-Caribbean [6.2, 19.4] Black-Other [0, 70.8] Arab [2.0, 58.1] Other [1.4, 46.8] Region of origin d UK [4.3, 7.0],0.001 Ireland [1.2, 17.3] India [16.4, 34.7] Pakistan [11.4, 28.9] Bangladesh [11.8, 38.9] Sri Lanka [7.2, 59.0] Nepal [0, 52.2] Afghanistan [29.7, 84.2] Africa [2.5, 16.4] Australasia [1.2, 47.8] Caribbean [3.9, 16.5] China [0, 19.5] Eastern Europe [0.5, 13.6] Middle East [2.8, 41.2] North America [1.4, 55.7] South or Central America [1.0, 37.4] South East or Pacific Asia [4.7, 36.7] Western Europe (excl. UK and Ireland) [4.6, 23.5] Mixed [0, 26.5] Mauritius/Seychelles [0, 36.9] Combination of region of origin d and country of birth with 11 groups born in UK; UK origin [4.4, 7.1],0.001 born in UK; Asia-IPB origin [6.3, 33.9] born in UK; Caribbean origin [3.3, 34.8] born in UK; Other origin [3.0, 20.2] born in India [18.7, 41.0] born in Pakistan [10.5, 30.7] born in Bangladesh [11.8, 41.3] born in Sri Lanka [7.2, 59.0] Continued 1373

7 McNulty et al. Table 2. Continued Factor (unweighted %) Prevalence [95% CI] (weighted %) a Test for a difference in prevalence between the groups of each factor OR adjusted for b (country of birth and region born in the UK) (aor) [95% CI] Test for the effect of each factor after adjustment for country of birth and region born in the UK born in Afghanistan [29.7, 84.2] born in the Middle East [4.7, 40.5] born in some other country [3.3, 8.5] Combination of region of origin d and country of birth with 8 groups born in UK; UK origin [4.4, 7.1],0.001 born in UK; Asia-IPB origin [6.3, 33.9] born in UK; Caribbean origin [3.3, 34.8] born in UK; Other origin [3.0, 20.2] born in India, Pakistan, Bangladesh or Sri Lanka [18.5, 32.9] born in Afghanistan [29.7, 84.2] born in the Middle East [4.7, 40.5] born in some other country [3.3, 8.5] Do you work in a healthcare setting? no [5.5, 8.0] yes [4.6, 12.6] 1.0 [0.6, 1.7] Type of healthcare worker nurse [2.1, 14.5] [0.3, 2.1] 0.25 care assistant [2.2, 29.9] 0.4 [0.1, 1.9] doctor [6.6, 40.5] 1.6 [0.6, 4.6] domestic [3.9, 61.1] 6.0 [1.1, 33.3] other work in healthcare (incl. unsp.) [2.3, 13.1] 1.1 [0.5, 2.3] not working in healthcare [5.5, 8.0] 1 Type of healthcare worker hands-on healthcare worker [4.4, 16.5] [0.4, 1.6] 0.53 hands-off healthcare worker (incl. unsp.) [2.9, 13.2] 1.4 [0.7, 2.6] not working in healthcare [5.5, 8.0] 1 Does your work involve contact with animals? no [5.6, 8.1] yes [2.8, 14.4] 1.2 [0.5, 2.7] Type of work involving contact with animals farm work (incl. meat prep.) [3.9, 22.6] [0.7, 4.5] 0.33 veterinary work [0, 24.7] 0 other work with animals (incl. unsp.) [0.4, 17.8] 0.7 [0.1, 5.4] not working with animals [5.6, 8.1]

8 CTX-M ESBL-producing Enterobacteriaceae prevalence JAC Have you been hospitalized in the past 12 months? no [5.4, 8.0] yes [4.4, 12.9] 1.1 [0.7, 1.7] Have you taken any antibiotics in the past 12 months? no [5.4, 8.6] yes [5.1, 8.9] 1.0 [0.7, 1.4] Have you taken any amoxicillin in the past 12 months? no [5.5, 8.4] yes [4.5, 9.7] 0.9 [0.6, 1.3] Have you taken any trimethoprim in the past 12 months? no [5.7, 8.2] yes [1.7, 15.0] 0.9 [0.3, 2.8] Have you taken any erythromycin in the past 12 months? no [5.6, 8.1] yes [3.6, 26.1] 1.3 [0.5, 3.5] Have you taken any clarithromycin in the past 12 months? no [5.7, 8.2] yes [1.6, 19.3] 1.3 [0.4, 5.0] Have you taken any co-amoxiclav in the past 12 months? no [5.8, 8.4] yes [0, 7.4] 0 Have you taken any ciprofloxacin in the past 12 months? no [5.6, 8.1] yes [2.5, 30.1] 3.0 [1.1, 8.1] Have you taken any cefalexin in the past 12 months? no [5.6, 8.1] yes [3.8, 29.3] 1.8 [0.6, 5.8] Have you taken any other antibiotics in the past 12 months? no [5.6, 8.2] yes [2.5, 16.7] 0.6 [0.3, 1.4] Have you taken co-amoxiclav, ciprofloxacin or cefalexin in the past 12 months? no [5.7, 8.3] yes [1.7, 9.4] 0.9 [0.4, 1.9] Do you currently have a urinary catheter? no [5.7, 8.2] yes [3.3, 49.3] 1.4 [0.4, 5.5] Do you regularly eat beef? no [5.8, 9.7] yes [5.1, 8.2] 1.0 [0.7, 1.4] Do you regularly eat pork/ham/bacon? no [8.5, 15.1], yes [4.6, 7.3] 0.8 [0.6, 1.2] Do you regularly eat lamb? no [4.8, 8.7] yes [5.6, 8.9] 1.2 [0.9, 1.7] Continued 1375

9 McNulty et al. Table 2. Continued Factor (unweighted %) Prevalence [95% CI] (weighted %) a Test for a difference in prevalence between the groups of each factor OR adjusted for b (country of birth and region born in the UK) (aor) [95% CI] Test for the effect of each factor after adjustment for country of birth and region born in the UK Do you regularly eat chicken? no [4.6, 12.1] yes [5.5, 8.1] 1.1 [0.7, 1.9] Do you regularly eat fish/seafood? no [6.0, 14.3] yes [5.1, 7.6] 0.9 [0.6, 1.4] Do you regularly eat salad products? no [5.2, 15.4] yes [5.5, 8.1] 0.6 [0.4, 1.1] Not regularly eating meat no [5.4, 8.0] yes [5.5, 15.1] 1.0 [0.6, 1.8] Not regularly eating meat, fish or seafood (vegetarian) no [5.5, 8.0] yes [5.0, 18.8] 0.8 [0.4, 1.6] In the past year, have you spent time in any country outside the UK? no [3.3, 6.3], ,0.001 yes [7.1, 10.8] 2.0 [1.5, 2.8] Have you been hospitalized abroad in the last year? no [5.5, 8.0], yes [13.1, 70.8] 3.6 [1.1, 12.2] Have you had diarrhoea abroad in the last year? no [5.4, 7.8] yes [6.1, 19.7] 1.8 [1.0, 3.2] Have you been abroad to India in the last year? no [5.1, 7.5], yes [22.6, 47.0] 3.2 [1.7, 6.2] Have you been abroad to Pakistan in the last year? no [5.5, 7.8], yes [24.8, 73.1] 3.6 [1.4, 9.1] Have you been abroad to Bangladesh in the last year? no [5.6, 8.1] yes [4.2, 50.1] 0.9 [0.2, 4.9] Have you been abroad to Sri Lanka in the last year? no [5.6, 8.0], yes [12.4, 75.6] 6.4 [1.3, 31.4] 1376

10 CTX-M ESBL-producing Enterobacteriaceae prevalence JAC Have you been abroad to Nepal in the last year? no [5.5, 7.9], yes [17.9, 98.0] 8.9 [0.6, 133] Have you been abroad to South Asia (India, Pakistan, Bangladesh, Sri Lanka or Nepal) in the last year? no [4.7, 7.1], ,0.001 yes [27.8, 50.5] 3.3 [2.0, 5.6] Have you been abroad to Afghanistan in the last year? no [5.6, 8.1] yes [1.3, 76.1] 3.5 [0.5, 25.2] Have you been abroad to Africa in the last year? no [5.1, 7.5], yes [9.4, 27.0] 2.2 [1.2, 3.7] Have you been abroad to Australasia in the last year? no [5.6, 8.1] yes [1.9, 31.5] 0.9 [0.2, 4.0] Have you been abroad to the Caribbean in the last year? no [5.6, 8.0] yes [3.3, 25.5] 1.4 [0.5, 3.9] Have you been abroad to China in the last year? no [5.5, 7.9] yes [7.1, 53.5] 2.9 [0.9, 9.1] Have you been abroad to Eastern Europe in the last year? no [5.7, 8.3] yes [3.3, 9.7] 0.9 [0.5, 1.6] Have you been abroad to the Middle East in the last year? no [5.6, 8.0] yes [4.5, 23.0] 1.7 [0.7, 3.8] Have you been abroad to North America in the last year? no [5.3, 7.7] yes [6.2, 19.8] 1.7 [1.0, 3.0] Have you been abroad to South or Central America in the last year? no [5.5, 7.9] yes [6.3, 46.4] 3.1 [1.1, 9.2] Have you been abroad to South East or Pacific Asia in the last year? no [5.3, 7.7] yes [9.0, 31.1] 3.3 [1.6, 6.5] Have you been abroad to a country in Western Europe (not incl. UK and Ireland) in the last year? no [5.1, 7.9] yes [5.5, 10.0] 1.3 [0.9, 1.8] Have you been abroad to Maldives, Mauritius or Seychelles in the last year? no [5.7, 8.1] yes [0, 18.5] 0 Have you been abroad to: Australia, the Caribbean, Eastern Europe, the Middle East, North America, Western Europe (not incl. UK and Ireland), Maldives, Mauritius or Seychelles in the last year? no [4.7, 7.7] yes [5.9, 9.8] 1.4 [1.0, 2.0] Continued 1377

11 McNulty et al. Table 2. Continued Factor (unweighted %) Prevalence [95% CI] (weighted %) a Test for a difference in prevalence between the groups of each factor OR adjusted for b (country of birth and region born in the UK) (aor) [95% CI] Test for the effect of each factor after adjustment for country of birth and region born in the UK Have you been abroad to: Africa, China, South or Central America, South East or Pacific Asia or Afghanistan in the last year? no [4.6, 6.8], ,0.001 yes [11.3, 23.7] 2.8 [1.8, 4.3] Number of adults living in respondent s household (incl. respondent) [3.8, 9.5] 0.02 (test for (test for [4.6, 7.5] trend: P " 0.06) 1.0 [0.6, 1.6] trend: P " 0.96) [5.3, 13.0] 1.1 [0.6, 1.8] [3.5, 10.6] 0.8 [0.4, 1.6] [9.8, 32.9] 1.7 [0.8, 3.5] 6! [3.0, 17.9] 0.9 [0.3, 2.1] Number of children aged 5 17 years living in respondent s household [5.6, 8.4] 0.18 (test for (test for [1.8, 7.0] trend: P " 0.77) 0.6 [0.3, 1.0] trend: P " 0.47) [5.1, 17.1] 1.1 [0.6, 1.9] 3! [3.4, 17.9] 0.8 [0.4, 1.6] Number of children aged 0 4 years living in respondent s household [5.2, 7.8] 0.12 (test for (test for [6.1, 15.6] trend: P " 0.28) 1.0 [0.6, 1.7] trend: P " 0.24) 2! [1.9, 10.4] 1.2 [0.6, 2.3] Number of children aged 0 17 years living in respondent s household [5.2, 8.1] 0.96 (test for (test for [4.2, 11.6] trend: P " 0.44) 0.8 [0.5, 1.3] trend: P " 0.96) [4.1, 12.0] 1.0 [0.6, 1.6] 3! [3.9, 15.1] 0.9 [0.5, 1.6] Number of people living in respondent s household [3.6, 9.4] 0.07 (test for (test for [4.8, 8.1] trend: P " 0.06) 1.0 [0.6, 1.7] trend: P " 1.00) [4.3, 11.8] 0.9 [0.5, 1.6] [2.5, 8.2] 0.7 [0.4, 1.4] [6.0, 15.1] 1.2 [0.7, 2.3] [6.1, 26.4] 1.0 [0.4, 2.2] [8.0, 38.7] 1.5 [0.6, 3.8] 8! [2.9, 14.0] 0.7 [0.3, 1.7] Do you have any cats living in your house or flat? no [5.7, 8.4] yes [4.1, 9.7] 1.1 [0.7, 1.6] Do you have any dogs living in your house or flat? no [5.9, 8.6] yes [3.0, 8.3] 1.0 [0.6, 1.7] 1378

12 CTX-M ESBL-producing Enterobacteriaceae prevalence JAC Do you have any rabbits living in your house or flat? no [5.4, 7.8] yes [1.9, 21.6] 0.8 [0.2, 3.0] Do you have any guinea pigs living in your house or flat? no [5.5, 7.9] yes [0.1, 6.4] 0.3 [0.04, 2.0] Do you have any hamsters living in your house or flat? no [5.5, 8.1] yes [0.3, 14.1] 0.5 [ ] Do you live in a nursing home, care home or residential home? no [5.7, 8.1] yes [0, 33.6] 0 Is there anyone in your household who works in a healthcare setting? no [5.5, 8.1] yes [4.0, 11.3] 0.9 [0.6, 1.5] Is there anyone in your household whose work involves contact with animals? no [5.7, 8.3] yes [2.0, 10.9] 1.1 [0.5, 2.7] Is there anyone in your household who has been hospitalized in the past year? no [5.7, 8.3] yes [3.2, 10.5] 0.8 [0.5, 1.4] Is there anyone in your household who has taken antibiotics in the past year? no [5.1, 8.1] yes [4.9, 8.9] 1.1 [0.8, 1.5] Is there anyone in your household who has spent time abroad in the last year? no [4.3, 7.3] ,0.001 yes [6.5, 10.5] 1.8 [1.3, 2.4] Housemate been to India in the last year no [5.4, 7.8], yes [14.5, 39.2] 2.4 [1.3, 4.6] Housemate been to Pakistan in the last year no [5.7, 8.1] yes [3.9, 45.5] 1.1 [0.3, 3.6] Housemate been to Bangladesh in the last year no [5.7, 8.2] yes [0, 30.8] 0 Housemate been to Sri Lanka in the last year no [5.6, 8.1] yes [5.5, 69.5] 3.7 [0.7, 19.6] Housemate been to the Indian subcontinent (India, Pakistan, Bangladesh or Sri Lanka) in the last year no [5.3, 7.7], yes [14.2, 34.1] 1.7 [1.0, 3.0] Housemate been to Afghanistan in the last year no [5.7, 8.2] yes [0, 84.2] 0 Housemate been to Africa in the last year no [5.5, 8.0] yes [5.0, 24.3] 1.5 [0.7, 3.0] Continued 1379

13 McNulty et al. Table 2. Continued Factor (unweighted %) Prevalence [95% CI] (weighted %) a Test for a difference in prevalence between the groups of each factor OR adjusted for b (country of birth and region born in the UK) (aor) [95% CI] Test for the effect of each factor after adjustment for country of birth and region born in the UK Housemate been to Australasia in the last year no [5.6, 8.0] yes [2.5, 44.0] 1.1 [0.3, 4.9] Housemate been to the Caribbean in the last year no [5.7, 8.1] yes [2.6, 26.9] 1.1 [0.3, 3.8] Housemate been to China in the last year no [5.6, 8.0] yes [5.6, 51.4] 2.6 [0.7, 9.9] Housemate been to Eastern Europe in the last year no [5.7, 8.2] yes [3.4, 12.1] 1.1 [0.6, 1.9] Housemate been to the Middle East in the last year no [5.6, 8.0] yes [7.5, 33.6] 2.5 [1.1, 5.6] Housemate been to North America in the last year no [5.6, 8.1] yes [4.2, 13.4] 1.6 [0.9, 2.8] Housemate been to South or Central America in the last year no [5.7, 8.1] yes [2.5, 23.4] 2.4 [0.7, 8.2] Housemate been to South East or Pacific Asia in the last year no [5.4, 7.8] yes [8.4, 34.1] 3.1 [1.4, 7.1] Housemate been to Western Europe (not incl. UK and Ireland) in the last year no [5.5, 8.3] yes [4.9, 9.7] 1.4 [0.9, 1.9] Housemate been to another region of the world in the last year no [5.7, 8.1] yes [1.1, 37.7] 0.8 [0.1, 5.5] Also shown are the ORs for potential risk factors for colonization after adjustment for the person s country of birth and the person s region born in the UK. a To estimate the prevalence of ESBLPE colonization for adults living in England in 2014 we used weights based on the 2011 national census and the number of eligible individuals at the selected practices. Weights for ethnic group, age group and sex were calculated for all individuals based on the census data alone. To estimate the prevalence for each GP practice and PCT we used weights based on the numbers of eligible individuals in each group at each practice. b Categories of this factor variable referred to in this table include: Born in the UK: UK origin; Born in the UK: India, Pakistan or Bangladesh origin; Born in the UK: Caribbean origin; Born in the UK: other origin; Born in India; Born in Pakistan; Born in Bangladesh; Born in Sri Lanka; Born in Afghanistan; Born in the Middle East; and Born in some other country. c Ethnic group was self-declared by each participant when completing the research questionnaire. d Region of origin/origin is derived from the self-declared ethnic group and country of birth. 1380

14 CTX-M ESBL-producing Enterobacteriaceae prevalence JAC Table 3. Final multivariate model for colonization with CTX-M ESBLPE Risk factor Categories Number of people exposed to the risk factor in the model (n " 2319) aor [95% CI]; P value Percentage PAF [95% CI] Percentage PAF [95% CI] Country of birth and region of origin a if born in the UK Born in the Indian subcontinent (India, Pakistan, Bangladesh or Sri Lanka) [3.0, 9.7];, [15.9, 30.9] 27.7 [19.5, 35.1] Born in Afghanistan [9.6, 218];, [1.3, 4.3] Born in the Middle East [1.3, 17.0]; [#0.4, 2.6] Born in the UK and of UK origin [0.8, 2.1]; [#7.1, 24.1] 15.3 [#3.5, 30.6] Born in the UK and of IPB origin [1.5, 9.2]; [0.1, 5.4] Born in the UK and of Caribbean origin [1.0, 10.9]; [#0.6, 3.3] Born in the UK and of some other origin or of mixed origin [0.7, 6.5]; [#1.0, 3.5] Compared with (Reference category): Born in some country other than UK, IPB, Sri Lanka, Afghanistan or the Middle East 464 Reference Reference Reference Travel abroad in the past year South Asia [1.8, 4.8];, [5.9, 7.8] 27.9 [16.0, 38.1] India Sri Lanka Pakistan Nepal Bangladesh Compared with (Reference category): No travel to South Asia 2186 Reference Reference Countries outside Asia with higher risk [1.7, 4.1];, [4.3, 15.1] Africa South East or Pacific Asia China Afghanistan South or Central America Compared with (Reference category): No travel to countries outside Asia with higher risk 2073 Reference Reference Other countries [0.9, 1.8]; [#3.5, 18.1] Australasia Mauritius The Caribbean Seychelles The Middle East Maldives North America Europe (excl. UK & Ireland) Compared with (Reference category): No travel to Other countries 1298 Reference Reference Domestic work in a healthcare setting Yes [1.3, 31]; [#0.2, 2.3] Compared with (Reference category): No 2311 Reference Reference a Region of origin/origin is derived from the self-declared ethnic group and country of birth. 1381

15 McNulty et al. England Overall Born UK : UK origin Born UK : Asia-IPB* origin Born UK : Caribbean origin Born UK : Other origin Born IPB or Sri Lanka Born India Born Pakistan Born Bangladesh Born Sri Lanka Born Afghanistan Born Middle East Born some other country (incl. Caribbean) % Prevalence of colonization with CTX-M ESBLPE *IPB = India, Pakistan or Bangladesh travelled abroad in the last year. Of the 199 E. coli, 87% (173/199) were ST131. ST131 was not significantly more common in CTX-M carriers who had spent time anywhere abroad in the last year, or in the Indian subcontinent (India, Pakistan, Bangladesh or Sri Lanka), compared with those who had not (spent time anywhere abroad 87% ST131 versus not spent time abroad 79%, P " 0.17; spent time abroad in the Indian subcontinent 91% ST131 versus not spent time in the Indian subcontinent 82%, P " 0.17). Risk factors for colonization with CTX-M ESBLPE Region of origin/origin is derived from the self-declared ethnic group and country of birth Figure 1. Prevalence of colonization with CTX-M ESBLPE by country of birth and region born in the UK (with 95% CI). Adults from the general population of England in Dotted line is the estimated 2014 prevalence in England. After adjusting for country of birth and region of origin (if born in the UK) we found no evidence for an independent association between CTX-M ESBLPE colonization and GP practice, age group, sex, overall antibiotic use in the past year or hospitalization in the past year (Table 2). Factors that remained significant after adjusting for country of birth and region of origin (if born in the UK) and were therefore considered for inclusion in the final model included: participant s travel abroad, or diarrhoea or hospitalization abroad in the last year; use of ciprofloxacin or co-amoxiclav; being a domestic healthcare worker; and housemates travel abroad (overall and by country) in the last year. When added to the final multivariable model, there was no strong evidence that colonization was independently associated with either taking antibiotics in the last year (aor 0.99, 95% CI , P " 0.95), hospitalization abroad in the last year (aor 2.7, 95% CI , P " 0.11, 3/12 colonized), diarrhoea while abroad in the last year (aor 1.1, 95% CI , P " 0.84, 16/137 colonized) or travel abroad by a participant s housemate in the past year (aor 1.4, P " 0.11, 103/1071 colonized). Use of ciprofloxacin remained a significant risk factor for ESBLPE-CTX-M in the final model (4/15 ESBLPE positive, aor 3.2, P " 0.03), whereas co-amoxiclav was protective for presence of ESBLPE-CTX-M (0/48 ESBLPE positive, aor 0, P " 0.006). Being born in the Indian subcontinent (India, Pakistan, Bangladesh or Sri Lanka) was the most important identified risk factor for faecal colonization (aor 5.4, 95% CI ), and we estimate it accounted for 23.8% (95% CI 15.9% 30.9%) of people colonized in England in 2014 (Table 3). Being born in Afghanistan (aor 46.0, 95% CI ) or the Middle East (aor 4.7, 95% CI ) were factors strongly associated with colonization, but being relatively rare we estimate them to have accounted for relatively few people colonized [2.8% (95% CI 1.3% 4.3%) and 1.1% (95% CI #0.4% to 2.6%), respectively]. We found no evidence when tested in the final model that birth in or travel to other countries including Eastern Europe increased risk of colonization (travel to Eastern Europe aor 0.8, P " 0.42; born in Eastern Europe aor 0.5, P " 0.38). There was no evidence that being born in the UK with a UK region of origin was a risk factor for colonization (aor 1.3, 95% CI , P " 0.24); but as there were so many participants in this group, we estimate it accounted for 9.9% (95% CI #7.1% to 24.1%) of people colonized. Being born in the UK with an IPB region of origin was strongly associated with colonization (aor 3.8, 95% CI ), and we estimate it accounted for 2.8% (95% CI 0.1% 5.4%) of people colonized. Being born in the UK with a Caribbean region of origin was almost as strongly associated with colonization as being born in the UK with an IPB region of origin (aor 3.4, 95% CI ), and we estimate it accounted for 1.4% (95% CI #0.6% to 3.3%) of people colonized. Travel to South Asia (India, Pakistan, Bangladesh, Sri Lanka or Nepal) in the last year was strongly associated with colonization 1382

16 CTX-M ESBL-producing Enterobacteriaceae prevalence JAC CTX-M-27: 6/27, 22% White 16/27, 59% Asian IPB 5/27, 19% Other ethnicity 16/27, 59% Travel abroad Group 8* 3, 1.4% CTX-M-15: 62/134, 46% White 46/134, 34% Asian IPB 26/134, 19% Other ethnicity 84/129, 65% Travel abroad (aor 2.9, 95% CI ), and we estimate it accounted for 12.1% (95% CI 5.9% 17.8%) of people colonized. Travel to Africa, China, South or Central America, South East or Pacific Asia or Afghanistan in the last year also increased the risk of colonization (aor 2.6, 95% CI ), and we estimate it accounted for 9.9% (95% CI 4.3% 15.1%) of people colonized. Travel to other countries in the last year put participants at a small increased chance of colonization (aor 1.3, 95% CI ; P " 0.15) and, being relatively common, we estimate it accounted for 7.9% (95% CI #3.5% to 18.1%) of people colonized. Working as a domestic in the healthcare setting was strongly associated with colonization (aor 6.2, 95% CI ), but, being relatively rare, we estimate it to have accounted for just 1.1% (95% CI #0.2% to 2.3%) of people colonized. Collectively all risk factors in the final multivariable model explained 60.4% (95% CI 40.0% 73.8%) of cases. Only 0.1% of participants (2/2430) were colonized with CPE; neither was born in the UK, and both had a history of travel to India in the past year. Discussion CTX-M-14: 19/23, 83% White 2/23, 9% Asian IPB 2/23, 9% Other ethnicity 14/23, 61% Travel abroad CTX-M-27 CTX-M-14 CTX-M-9 New? 27 (13%) 23 (11%) 1 (0.5%) 1 (0.5%) Not Typed 1 (0.5%) Group 9 53, 24% The 7.3% estimate for the prevalence of faecal colonization with CTX-M ESBLPE in adults living in England in 2014, and the high estimated prevalence in those born in South Asia (India, Pakistan, Bangladesh or Sri Lanka) and in those travelling to certain areas including South Asia, is of concern and has implications for empirical antimicrobial prescribing for suspected infections caused by Enterobacteriaceae and infection prevention and control within healthcare in England and beyond. The significantly higher Group 1 161, 77% CTX-M-15 CTX-M-1 CTX-M-55 CTX-M-3 CTX-M-32 New? Not Typed 134 (66%) 9 (4.4%) 8 (3.9%) 3 (1.5%) 2 (1%) 1 (0.5%) As each isolate could have more than one genotype, the relative frequencies for the different genotypes sum to more than 100% of individuals as nine individual isolates carried both CTX-M group 1 and group 9 genes *The three isolates in group 8 were not sequenced Figure 2. Relative frequency of the genotypes from 208 individuals, characterizations of these genotypes and risk factors for them. 4 (2%) estimated prevalence (15.7%) in the 52 participants born in the UK with an IPB region of origin, compared with those born in the UK with a UK region of origin (1459 participants, estimated prevalence 5.6%), is interesting; the higher estimated prevalence may be due to acquisition during repeated travel to their country of origin, or from visits by family and friends to or from their country of origin, during the last year or more than 1 year ago, or acquisition from relatives in the home. 22 Previous studies in the UK The only previous faecal colonization study of ESBLPE in the UK (in 2010) showed that Middle Eastern or South Asian (India, Pakistan, Bangladesh, Sri Lanka or Nepal) patients being investigated for gastrointestinal infections had a significantly higher prevalence than Europeans. 11 Although UK studies estimating ESBLPE infection rates in hospitalized patients or patients with urine infections (UTIs) have suggested (similar to this present study) that rates of ESBLPE infection vary widely between different areas of the UK 23,24 they have not investigated other risk factors. Previous studies of a general population in Northern Europe A 2011 postal study in urban Amsterdam estimated that the overall prevalence of ESBLPE faecal colonization was 8.6% and travel to Asia or Africa (aor ) in the last year increased the risk. Unlike the present or other studies, they found antibiotic use in the last year and travel to North America (aor 2.7) were also 1383